DE69125842T2 - INACTIVATED MYCOPLASMA HYPOPNEUMONIAE BACTERIN AND METHOD FOR USE THEREOF - Google Patents

Abstract

This invention provides a bacterin comprising a virulent Mycoplasma hyopneumoniae isolate, inactivated with binary ethyleneimine, in an amount effective to immunize a swine against infection by Mycoplasma hyopneumoniae and a suitable physiologically acceptable carrier. The invention also provides a method of producing this bacterin. The invention further provides a method of inactivating a virulent strain of Mycoplasma hyopneumoniae by contacting the Mycoplasma hyopneumoniae with binary ethyleneimine. Finally, the invention also provides a method of immunizing swine against infection by Mycoplasma hyopneumoniae comprising administering to the swine a dose of the bacterin so as to immunize the swine.

Description

State of the art

Mycoplasma hyopneumoniae is a ubiquitous respiratory pathogen of swine. It infects the respiratory tract of pigs, colonizing the trachea, bronchi, and bronchioles. Mycoplasma produces a ciliostatic factor that causes the cilia lining the airways to stop beating. Eventually, the cilia degenerate, leaving the pig susceptible to infection with more dangerous secondary pathogenic organisms. The disease caused by Mycoplasma hyopneumoniae, enzootic pneumonia, is a chronic, non-lethal disease that affects pigs of all ages (R. F. Ross, Mycoplasmal Diseases, pp. 436-444, in A. D. Lamen et al., (eds.), Diseases of Swine, Iowa State University Press, 1986). Infected pigs show only mild symptoms of cough and fever. However, the economic impact of the disease is significant. The disease is one of the leading causes of disease-related losses in pigs (Whittlestone, pp. 133-176, in Tully and Whitcomb (eds.), The Mycoplasma, Vol. 2: Human and Animal Mycoplasmas, New York, Academic Press, (1979)). The clinical picture is generally one of failure to gain weight, failure to develop properly, and illness. In addition, affected pigs are often susceptible to secondary infection by opportunistic organisms (Burch, Fig America, pp. 26-27, December 1982).

A great deal of effort has been put into researching vaccines that can protect pigs against mycoplasma pneumonia. However, no effective vaccines have been found to date, so the disease is still very widespread.

Several investigators have described vaccines containing recombinantly produced surface antigens of Mycoplasma hyopneumoniae, Schaller et al., U.S. Patent No. 4,894,332, issued January 16, 1990; European Patent Publication No. 283,840, published September 28, 1988.

PCT Publication No. WO 86/00019, published on January 3, 1986, describes a Mycoplasma hyopneumoniae vaccine comprising exclusively Mycoplasma hyopneumoniae plasma membranes, free from other cellular components.

Etheridge et al., Res. Vet. Sci. 33 (1982), 188, found that intravenous, subcutaneous or intraperitoneal administration of a live vaccine provided only incomplete protection against lung colonization by Mycoplasma hyopneumoniae.

Kristensen et al., Am. J. Vet. Res. 42 (1981), 784, found no protection against mycoplasma pneumonia following injection of heat-inactivated Mycoplasma hyopneumoniae in pigs.

Ross et al., Am. J. Vet. Res. 45 (1984), 1899, found that when Mycoplasma hyopneumoniae extracts prepared by a freeze-thaw procedure were used to immunize pigs, only variable protection was provided, with increased lesion development in some cases observed in the immunized pigs. These investigators also studied a whole-cell vaccine prepared by formalin inactivation. Formalin inactivation significantly interfered with the protective immunogenicity of Mycoplasma hyopneumoniae and this vaccine was ineffective.

Yoshioka et al., U.S. Patent No. 3,917,819, issued November 4, 1975, describe various killed mycoplasma vaccines comprising formalin-inactivated mycoplasmas, including an inactivated vaccine for mycoplasma hyopneumoniae. However, this vaccine was produced by inactivation with formalin.

G. Christianson et al. (Chemical Abstracts, 1980, Abstract No. 19975p) describe that binary ethyleneimine was not always successful in inactivating mycoplasma contaminants in sera.

Thus, previous attempts with inactivated Mycoplasma hyopneumoniae vaccines were unsuccessful, presumably because the conditions used, i.e., heat treatment, formalin treatment or freeze-thawing, destroyed the protective antigenic potential of the organism.

Accordingly, no effective whole-cell vaccine containing inactivated virulent Mycoplasma hyopneumoniae has yet been developed to protect pigs against mycoplasma pneumonia.

This invention provides a whole cell Mycoplasma hyopneumoniae vaccine or bacteria comprising Mycoplasma hyopneumoniae inactivated by treatment with binary ethyleneimine. Contrary to other inactivation techniques, this method does not adversely affect the protective immunogenicity of the organism, thus allowing it to be formulated as an effective bacterin.

Summary of the invention

This invention provides a bacterin comprising a binary ethyleneimine-inactivated virulent Mycoplasma hyopneumoniae isolate in an amount effective to immunize a pig against infection by Mycoplasma hyopneumoniae and a suitable physiologically acceptable carrier.

This invention also provides a process for producing a bacterin comprising growing a virulent Mycoplasma hyopneumoniae isolate in culture on a suitable medium; treating the virulent Mycoplasma hyopneumoniae grown in this way with binary ethyleneimine so as to inactivate the Mycoplasma hyopneumoniae; concentrating the resulting inactivated Mycoplasma hyopneumoniae; recovering the resulting concentrated inactivated Mycoplasma hyopneumoniae; and Mixing the concentrated inactivated Mycoplasma hyopneumoniae thus obtained with a suitable physiologically acceptable carrier, thereby formulating the bacterin.

This invention also provides a method for inactivating a virulent strain of Mycoplasma hyopneumoniae comprising contacting a culture of Mycoplasma hyopneumoniae with binary ethyleneimine at a concentration of about 4 mM; incubating the culture under conditions effective to inactivate the Mycoplasma hyopneumoniae; and neutralizing the binary ethyleneimine in the culture by adding sodium thiosulfate at an effective neutralizing concentration.

This invention further provides a method of immunizing a pig against infection by Mycoplasma hyopneumoniae comprising administering to the pig a dose of a bacterin according to the present invention such that the pig is immunized against Mycoplasma hyopneumoniae infection.

Short description of the characters

Figure 1: ELISA results comparing the relative antibody titres induced in pigs by a bacterin produced by inactivation of Mycoplasma hyopneumoniae with binary ethyleneimine with those induced by a bacterin produced by inactivation with formalin.

Figure 2: Results of a study on the minimum protective dose of binary ethyleneimine-inactivated bacterin. Serum samples were taken from each pig in the test at 1 (PV), 3 (V1), 4 (V2), 5 (PCH1) and 7 (PCH3) weeks of age and antibody titres were tested by ELISA.

Detailed description of the invention

The present invention provides a bacterin comprising a binary ethyleneimine-inactivated virulent Mycoplasma hyopneumoniae isolate in an amount sufficient to immunize a pig against infection by Mycoplasma hyopneumoniae, and a suitable physiologically acceptable carrier. In a preferred embodiment of the present invention, the Mycoplasma hyopneumoniae isolate of the bacterin comprises the isolate designated 1002 (ATCC Accession No. 55088).

Although the present invention is described below with respect to the Mycoplasma hyopneumoniae isolate designated 1002 (ATCC Accession No. 55088), it is to be understood that any virulent Mycoplasma hyopneumoniae isolate inactivated with binary ethyleneimine can be formulated as an effective bacterin. Numerous virulent Mycoplasma hyopneumoniae isolates are known to those skilled in the art and are available from various sources, including the American Type Culture Collection. Finally, isolate 1002 has been deposited for the purpose of patent proceedings with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, in accordance with and in fulfillment of the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

The bacterin further comprises a suitable physiologically acceptable carrier. Many different suitable carriers are known to the person skilled in the art, who can select a specific carrier from them. The suitable physiologically acceptable carriers mentioned merely as examples include distilled or deionized water, saline solution or mineral oil.

In a preferred embodiment herein, the bacterin further comprises an effective amount of an adjuvant. An "adjuvant" as used herein is an enhancer of the immune response. Adjuvants may include poisons, irritants, and generally substances that boost the immune response of the animal receiving the injection. In a preferred embodiment of the invention herein, the adjuvant comprises a polymer of acrylic acid, i.e., a homopolymer. A commercially available example of such a homopolymer of acrylic acid is Carbopol 941 (BF Goodrich Co., Cleveland, Ohio). The chemical formula of Carbopol is (CH₂CHOOOH)n. The effective amount of an any specific adjuvant can be readily determined and in this way the effect of the adjuvant in enhancing the immune response of an animal vaccinated with the bacterin can be optimized. In general, an effective amount of acrylic acid polymer suitable as an adjuvant is about 0.2% (w/v) of the bacterin.

The bacterin of the invention comprises the binary ethyleneimine-inactivated virulent Mycoplasma hyopneumoniae isolate in an amount effective to immunize a pig. "Effectively immunizing" a pig against Mycoplasma hyopneumoniae infection means that the bacterin prevents or at least alleviates mycoplasma pneumonia. In any case, one of skill in the art can readily determine, for any specific combination of isolate and carrier, in the presence or absence of an adjuvant, the particular amount of binary ethyleneimine-inactivated Mycoplasma hyopneumoniae that is effective to immunize a pig. In the presently preferred embodiment of the invention, the effective amount is at least about 10⁹ Mycoplasma hyopneumoniae DNA cell equivalents per milliliter of bacterin (see Materials and Methods, below, for explanation of "DNA cell equivalents").

This invention also provides a process for producing a bacterin comprising growing a virulent isolate of Mycoplasma hyopneumoniae in culture in a suitable medium; treating the virulent Mycoplasma hyopneumoniae thus grown with binary ethyleneimine so as to inactivate the Mycoplasma hyopneumoniae; concentrating the resulting inactivated Mycoplasma hyopneumoniae; recovering the resulting concentrated inactivated Mycoplasma hyopneumoniae; and mixing the concentrated inactivated Mycoplasma hyopneumoniae thus recovered with a suitable physiologically acceptable carrier, thereby formulating the bacterin. In a preferred embodiment of the invention, the Mycoplasma hyopneumoniae isolate designated 1002 (ATCC- Accession number 55088) was used to formulate the bacterin.

In particular, a virulent isolate of Mycoplasma hyopneumoniae is grown in culture in a suitable medium containing Mycoplasma base broth, yeast extract, glucose, L-cysteine, ampicillin, thallium acetate, porcine serum and deionized water. Such a medium, which is preferred here, is described in more detail below.

The specific conditions under which the isolate is grown may depend on the actual composition of the medium and the specific isolate being grown. Typically, however, the isolate is grown for about 48 hours to about 144 hours, measured from the time of incubation to the time of harvest.

The virulent Mycoplasma hyopneumoniae isolate grown in this manner is then treated with binary ethyleneimine, which inactivates the Mycoplasma hyopneumoniae. To this end, the isolate culture can be contacted with binary ethyleneimine at a concentration of about 1 to about 10 mM, e.g., about 4 mM. The culture is then incubated under conditions effective to inactivate the Mycoplasma hyopneumoniae, e.g., at about 37°C for at least about 12 hours. The binary ethyleneimine in the culture is then neutralized by the addition of sodium thiosulfate at an effective neutralizing concentration, e.g., a concentration of about 10 to about 20 mM, such as about 16 mM.

In one embodiment of the invention, the binary ethyleneimine used to contact and inactivate the Mycoplasma hyopneumoniae is generated in situ by adding L-bromoethylamine hydrobromide to the culture in an amount that, after conversion to the binary ethyleneimine, results in the establishment of the desired inactivating concentration, e.g., about 4 mM.

The resulting inactivated Mycoplasma hyopneumoniae is then concentrated. Various methods for concentrating such organisms are known to those skilled in the art. For example, the organisms can be concentrated by centrifugation, e.g. ultracentrifugation, or by filtration, e.g. ultrafiltration.

The resulting concentrated, inactivated Mycoplasma hyopneumoniae is then recovered, again using methods known to those skilled in the art.

Finally, the resulting concentrated inactivated Mycoplasma hyopneumoniae thus obtained is mixed with a suitable physiologically acceptable carrier, thereby formulating the bacterin. Most preferably, the mixture may further comprise an effective amount of EDTA, e.g., about 0.05% to about 0.20%, particularly at least about 0.07% (w/v) of the bacterin, or an effective concentration of thimerosal, e.g., about 0.005% to about 0.05%, particularly about 0.01% (w/v) of the bacterin.

The bacterin can also be prepared by any modification of the above procedure. For example, the Mycoplasma hyopneumoniae can be concentrated before rather than after inactivation. Also, the preparation of the bacterin can be carried out by repeating the inactivation step both before and after the concentration step.

The concentrated binary ethyleneimine inactivated Mycoplasma hyopneumoniae is formulated by adding a suitable carrier to the bacterin. A diluent, colorant or preservative may also be added. A chelating agent such as EDTA may be added, preferably to a final concentration of at least about 0.07% (w/v). Diluents such as saline or other carriers known to those skilled in the art may be used to dilute the inactivated Mycoplasma hyopneumoniae in the formulation of the bacterin. Preservatives may also be added. In one embodiment of the invention, thimerosal is added as a preservative to a final concentration of at least about 0.01% (w/v).

This invention also provides a method for inactivating a virulent strain of Mycoplasma hyopneumoniae comprising contacting a culture of Mycoplasma hyopneumoniae with binary ethyleneimine at a concentration of about 4 mM; incubating the culture under conditions effective to inactivate the Mycoplasma hyopneumoniae are effective, such as an effective time, eg about 12 hours, at an effective temperature, eg about 37ºC; and neutralizing the binary ethyleneimine in the culture by adding sodium thiosulfate to the culture at an effective neutralizing concentration, eg about four times the concentration of the binary ethyleneimine, ie about 16 mM when the concentration of the binary ethyleneimine is about 4 mM. Also, the binary ethyleneimine can be generated in situ as indicated above by adding L-bromoethylamine hydrobromide to the culture.

This invention also relates to a method for immunizing a pig against infection by Mycoplasma hyopneumoniae, comprising administering to the pig at least one dose of the bacterin, thereby immunizing the pig against Mycoplasma hyopneumoniae infection. The bacterin can in principle be administered by various routes. However, in the preferred embodiment of the invention, the bacterin is administered intramuscularly. The dose of the bacterin preferably comprises about 2 ml of the bacterin, wherein per ml there are about 10⁹ Mycoplasma hyopneumoniae DNA cell equivalents. The bacterin is best administered to the pig twice; once about one week and once about three weeks after the pig is born.

A bacterin according to the present invention for administration by injection comprises inactivated virulent Mycoplasma hyopneumoniae suspended in an inert physiologically acceptable liquid diluent.

Suitable diluents include water, saline, or any other diluent known to those skilled in the art. Advantageously, Mycoplasma hyopneumoniae is suspended in a sterile diluent under aseptic conditions.

The bacterin for injection can be made isotonic by any conventional technique, eg by dialysis against a saline solution that is isotonic with the blood of the pig. Any saline solution suitable for the Suitable for use as an injection medium, it can be used to make the bacterin isotonic.

Furthermore, the bacterin can be prepared either as a concentrated composition that must be diluted before use or as a ready-to-use composition. In the latter case, the concentration of inactivated virulent Mycoplasma hyopneumoniae is at least about 109 DNA cell equivalents per milliliter.

The binary ethyleneimine-inactivated Mycoplasma hyopneumoniae of the present invention can also be used as a component of a vaccine containing one or more other active ingredients, i.e. antigenic substances that can induce a protective immune response against Mycoplasma hyopneumoniae or against other disease-causing agents.

material and methods

The microorganism used to produce the bacterin is a virulent Mycoplasma hyopneumoniae isolate designated 1002 and deposited under ATCC accession number 55088. This strain, originally isolated from an infected lung by culturing a 10-2 dilution of a lung homogenate in mycoplasma broth, was passaged seven times in broth to prepare the master seed (“X”).

The identity of the stock preculture ("X") was determined by plating it on Mycoplasma Broth Agar and examining growth for typical Mycoplasma hyopneumoniae morphology. Mycoplasma hyopneumoniae strain J obtained from NVSL was used as a reference for comparison. The identity of the stock preculture ("X") was further confirmed by comparing it to the reference strains Mycoplasma hyopneumoniae strain J and other related Mycoplasma species by SDS-PAGE and immunoblot.

The stock preculture ("X") is the seventh passage of the isolate designated 1002. The preculture for production is the "X+2" passage. Preculture batches range from "X+3" to "X+6". Production cultures range from "X+6" to "X+7".

The pre-cultures and production cultures are propagated in the following medium:

1. Mycoplasma base broth (commercially available) 16,800 g

2. Yeast extract (commercially available) 1,000 g

3. Glucose 5,000 g

4. L-cysteine hydrochloride 0.100 g

5. Ampicillin 0.250 g

6. Thallium acetate, USP 0.250 g

7. Phenol red (indicator dye) (for use in pre-culture medium) 0.020 g

8. Normal sterile swine serum (may be heat inactivated in preculture medium at 58.5ºC for 30 minutes before use) 100,000 ml

9. Deionized water to 1000,000 ml

After thoroughly dissolving the components, the pH is adjusted to 7.8 ± 0.4 with NaOH. The solution is filtered into sterile containers by filtration through either a sterile 0.3 µm (micron) "Depth" filter, a sterile 0.3 µm (micron) membrane filter, or a sterile 0.2 µm (micron) filter cartridge. The "Depth" filters, membrane filters or filter cartridges and the holders are sterilized at 121ºC ± 2ºC for at least 30 minutes before use.

The basic medium may be sterilized without the glucose, the l-cysteine hydrochloride, ampicillin and porcine serum for at least 30 minutes at 121ºC ± 2ºC before use. The l-cysteine hydrochloride and ampicillin are sterilized by filtration through a 0.2 µm (micron) filter and added to the basic medium under aseptic conditions.

The glucose solution is autoclaved separately as a 30 ºC (w/v) solution for 30 minutes at 121ºC ± 2ºC. Normal sterile porcine serum is added under aseptic conditions. The pH of the complete medium is adjusted to 7.8 ± 0.4 using sterile 5 N NaOH solution. The preculture medium can be stored at 2 to 7ºC for up to 30 days. The autoclaved production culture medium can be stored at 2 to 7ºC for up to 10 days.

Pre-cultures are grown in 250- to 4000-mL polycarbonate or glass flasks, 12-liter glass bottles, 28- and 200-liter fermentation vessels containing 30- to 80-percent medium. Production cultures are grown in 200- to 3000-liter fermentation vessels containing 30- to 80-percent medium. The stock pre-culture ("X") is maintained in 100-percent medium at -20ºC or colder. The production pre-culture ("X+2") is maintained in 90-percent medium and 10-percent glycerol at -20ºC or colder.

The suspensions are prepared for addition as a pre-culture or for inoculation by rapidly thawing the frozen pre-cultures ("X" or "X+2") and inoculating them into the medium at a rate of 5 to 15% (vol/vol). The pre-cultures are serially transferred into the medium at a rate of 5 to 15% (vol/vol).

Pre-culture and production media are inoculated using the following technique. Frozen production pre-culture ("X+2") is used to inoculate medium in 250 mL flasks at a rate of 5 to 15% (vol/vol) to give the "X+3" passage. Subsequent pre-cultures ("X+4" to "X+9") are prepared by serially diluting each at a rate of 5 to 15% (vol/vol) into the appropriate containers. Cultures in 250 mL flasks can be used to inoculate 4000 mL flasks at a rate of 5 to 15% (vol/vol). Cultures in 4000 mL flasks can be used to inoculate 12 L bottles at a rate of 5 to 15% (vol/vol). Cultures in 12 liter bottles can be used to inoculate 28 to 200 liter fermentation vessels at a rate of 5 to 15% (vol/vol). Cultures in 28 to 200 liter fermentation vessels can be used to inoculate 200 to 3000 liter production fermentation vessels at a rate of 5 to 15% (vol/vol). Pre-cultures and production cultures are incubated for 48 to 120 hours at 37ºC ± 2ºC. All cultures are incubated with agitation during the growing period. The fermentation vessels can be aerated by supplying sterile air. The pH of the pre-cultures and the production cultures can be maintained at 7.4 ± 0.4 by adding a sterile NaOH solution. If necessary, a sterile 30% glucose solution can be added to the production culture.

Growth characteristics are determined by macroscopic observation. Characteristic growth is characterized by a decrease in the pH of the medium (pre-culture) or by an increased application of NaOH, which maintains the pH of the medium at a certain value (production cultures).

Growth intensity is determined by DNA fluorometry as follows: four 1.5 ml culture aliquots are placed in microfuge tubes. Samples are centrifuged at 12,000 xg for 10 minutes. The supernatants are completely removed and the pellets are resuspended in 120 µl of the following solution: 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1% (w/v) sodium dodecyl sulfate (TNES). The resuspended pellets are vigorously vortexed for 10 seconds. Ten µl of the resuspended pellets are mixed in a glass cuvette with 2 ml of the following Hoechst dye solution: 0.4 µg/ml in 10 mM Tris (pH 7.4), 150 mM NaCl and 1 mM EDTA (TNE). The cuvette is then placed in a fluorometer that has been previously calibrated using DNA standards. The intensity of the fluorescence is proportional to the amount of DNA present. The number of Mycoplasma hyopneumoniae DNA cell equivalents (MHDCE) in the culture is calculated using the following formula:

Before harvesting, the growth characteristics and strength of the production crops are determined by means of microscopic examination, and it is also determined whether contamination is present.

Before inactivation, the pH of the production cultures is adjusted to 8.2 ± 0.2 by adding sterile 5 N NaOH solution.

For inactivation, L-bromoethylamine hydrobromide (BEA) is added directly to the production culture in the fermentation vessel to a final concentration of not less than 4 mM.

The culture is kept with agitation for 12 to 24 hours at 37ºC ± 2ºC.

The mycoplasmas are inactivated by contacting them with binary ethyleneimine. The binary ethyleneimine (BEI) (primary inactivating agent) generated in situ during the incubation of L-bromoethylamine hydrobromide (BEA) in the production culture is neutralized by adding sodium thiosulfate directly to the inactivated production culture in the fermentation vessel to a final molar concentration of not less than four times the molar concentration of the binary ethyleneimine (BEI).

The culture is kept with movement for 12 to 24 hours at 37ºC ± 2ºC. The shortest and longest times from inoculation to harvest are 48 and 144 hours respectively.

For harvesting, the substances from the production vessel are combined in a sterile container. The harvest fluids are concentrated by centrifugation and/or ultrafiltration to 10% ± 5% of the original volume and can be diafiltered using sterile saline. The concentrate can be inactivated again by adding L-bromoethylamine hydrobromide (BEA) to a final concentration of not less than 4 mM. The concentrate is incubated with stirring for 12 to 24 hours at 37ºC ± 2ºC. The binary ethyleneimine (BEI) produced during inactivation is then neutralized by adding sodium thiosulfate to to a final molar concentration of not less than four times the molar concentration of binary ethyleneimine (BEI). The concentrate is incubated with stirring for 12 to 24 hours at 37ºC ± 2ºC. Thimerosal and ethylenediaminetetraacetic acid (EDTA) are added to a final concentration of 0.01% and 0.07%, respectively.

Only those production cultures that show characteristic growth are harvested. Before harvesting, each production vessel is subjected to a Gram stain.

The bacterin is preserved by adding thimerosal and ethylenediaminetetraacetic acid (EDTA) with not more than 0.01% and 0.07%, respectively.

As an adjuvant, Carbopol (B. F. Goodrich Co.) is added to the bacterin to a final concentration of 0.2% (w/v).

Sterile saline solution can be used as a diluent. The bacterin can be mixed with Red Dye FD&C 40 to a final concentration of 15 mg per liter.

The concentration of Mycoplasma cells in the final product is not less than 2 x 10⁹ Mycoplasma hyopneumoniae DNA cell equivalents per 2.0 ml dose as determined by the DNA fluorometry assay.

Suitable mycoplasma concentrates are added to the adjuvant, preservative, dye and diluent under aseptic conditions in a sterile container equipped with a stirrer and mixed for at least 30 minutes.

The present invention is described in more detail in the following examples. However, the scope of the claims should not be limited in any way by the examples.

example 1

The virulence of the following four different strains of Mycoplasma hyopneumoniae (MHP) was investigated: Mhp strain 1002, Mhp strain 1005-NB 12, Mhp strain 1003-J, Mhp strain 1004- 11 and Mhp strain 1006-415.

The virulence of the strains tested was determined by the lesions on the lungs of the exposed ("exposed") pigs. Based on this criterion, M. hyopneumoniae 1002 was determined to be virulent and this strain was used for the immunity test and for the production of the bacterin.

29 three-week-old pigs tested for pseudorabies were divided into four groups of eight, eight, seven and six animals. The pigs were reared in isolation until six weeks of age, at which time they were challenged with the virulent strain.

A low-passage stock culture of each of the four strains of Mycoplasma hyopneumoniae was thawed and a 10% inoculum of each strain was added to complete medium for pleuro-pneumonia-like organisms (PPLO) (see page 19). The cultures were incubated for 12 days at 37ºC.

Six ml of the appropriate infectious culture was administered into each nostril using a 10 ml syringe with a 1 1/2 inch rubber tube attached. To facilitate inhalation, a plastic bag was placed over the proboscis for a short time. This exposure was repeated on the following three days.

The infectious material was quantified by determining the color changing units (CCU).

All pigs were euthanized and necropsied three weeks after the first exposure. The lungs were removed and examined for characteristic M. hyopneumoniae lesions.

The areas of a lung with typical M. hyopneumoniae lesions were excised and placed in suitable sterile containers and frozen at -70ºC. At a later time, attempts were made to isolate M. hyopneumoniae from these organ samples.

The following results are summarized in Tables 1 and 2. Four different strains of M. hyopneumoniae were used in this study. The total dose ranged from 9.0 x 10⁷ to 9.0 x 10¹⁰ CCU/pig. The percentage of animals with lung lesions per group ranged from 17 to 50%. In all pigs, lung lesions were restricted to the cardiac lobes and apices and were characteristic of M. hyopneumoniae.

There appeared to be no relationship between dosage (CCU per pig) and the extent of lung lesions. M. hyopneumoniae 1002 and Mhp 1005-NB12 appeared to be the most virulent. However, M. hyopneumoniae 1002 grew faster in our PPLO medium and reached a higher CCU level.

Attempts to isolate the strains with which the animals had been exposed from the lung samples taken were successful. The cultures were examined under a phase contrast microscope and most likely contained M. hyopneumoniae.

M. hyopneumoniae 1002 was the strain of choice for further work, taking into account lung involvement, growth in PPLO medium, and previous studies using the isolate. Table 1 Lung lesions resulting from intranasal administration of four different strains of Mycoplasma hyopneumoniae Table 2 Lung lesions caused by four different strains of M. hyopneumoniae Table 2

PPLO complete medium I. Components A. PPLO broth w/o crystal violet (800 ml)

a. 16.8 g Difco PPLO w/o Crystal Violet dissolved in deionized water to 800 mL;

b. Mix with 500 g Amberlite IRA 400 on a magnetic stirrer, 2 hours at room temperature;

c. Filter off Amberlite using a paper filter;

B. Thallium acetate (2.5 ml)

a. 0.25 g thallium acetate dissolved in a total volume of 2.5 ml deionized water;

C. Phenol red (0.5 ml)

0.02 g Phenol red, dissolved in 0.5 ml deionized water;

D. L-cysteine hydrochloride (1 ml)

a. 0.1 g L-cysteine hydrochloride dissolved in 1.0 ml deionized water (prepare on the day of use);

E. Ampicillin (2.5 ml)

a. 0.25 g ampicillin dissolved in 2.5 ml deionized water (prepare on the day of use);

F. Glucose (50 ml)

a. 10 g glucose dissolved in a total volume of 50 ml deionized water;

G. Yeast extract (50 ml)

a. 25 g yeast (Fleischman Co.) dissolved in 150 ml deionized water, stirring rapidly on a magnetic stirrer;

b. 0.81 ml 10 N NaOH, slowly added to the yeast mixture;

c. Autoclave at 15 psi, 15 minutes;

d. Store overnight at 4ºC;

e. Centrifugation, 30 minutes at 8000 rpm;

f. Pouring off, collecting and measuring the supernatant;

g. Add 2 ml of 1 N HCl per 100 ml of supernatant;

h. Centrifugation, one hour at 10,000 rpm;

i. Drain and collect the supernatant;

j. Filter the supernatant first through a 0.8 µm filter, then through a 0.45 µm filter and finally through a 0.2 µm filter;

k. Store at 4ºC;

H. Gibco Pig Serum (100 ml)

a. Heat inactivation, 30 minutes at 56ºC;

II. Association of components

a. combination of the components in the quantities shown in brackets;

b. Filtration first through a 3.0 µm filter, then through a 0.45 µm filter and finally through a 0.2 µm filter;

c. Incubate overnight at 37ºC to check purity;

d. Store at 4ºC.

Color changing units Materials and equipment:

MHP complete medium

12 12x75mm plastic tubes with snap caps Pipette or Cornwall syringe

Culture

Procedure:

1) Add 1.8 ml of medium to all 12 tubes;

2) Add 0.2 ml of culture to the first tube and dilute 10-fold to tube 11; discard 0.2 ml of culture from tube 11;

3) Tube 12 contains no culture and is intended as a control;

4) securely cap the tubes and incubate for 14 days at 37ºC;

5) Examine for color change to determine titer; on day 14, record the highest dilution at which the color changed from red to yellow and calculate the CCU/ml as follows: CCU/ml = Highest dilution with color change x 0.2/2.0

Example 2

The binary ethyleneimine (BEI)-inactivated and the formalin-inactivated Mycoplasma hyopneumoniae bacterin were tested for their ability to protect pigs against infection with virulent M. hyopneumoniae.

The formalin-inactivated bacterin gave significantly higher ELISA titres than the BEI-inactivated bacterin. However, only the BEI-inactivated bacterin provided significant protection against infection. These results suggest that local secretory antibodies and/or cell-mediated immunity play a more important role in the immune response to M. hyopneumoniae than circulating antibodies. The evaluation of these results suggests that further studies should be conducted with BEI-inactivated bacterin.

The pigs used in this study were obtained from Ionia Pigs, Incorporated (Ionia, Iowa).

Mycoplasma hyopneumoniae strain 1002, originally designated P 5723-3 (Dr. Charles Armstrong, Purdue University) was sequentially passaged in medium as described above to passage X+7, using 10% inoculum for each passage. Each passage was incubated for 72 to 80 hours at 37ºC with vortexing at 75 rpm.

At the end of the incubation of the X+7 passage culture, the strength of growth and the concentration of mycoplasmas were determined visually by the coloring of the culture medium and by phase contrast microscopy, respectively. A sample was taken to determine the number of microorganisms present, using the quantification method using "color changing units" (CCU).

Inactivation - Formalin: Formalin was added to 400 ml of an X+7 culture to a final concentration of 0.2%. The cell suspension was vortexed at 75 rpm at 37ºC for 24 hours.

Inactivation - binary ethyleneimine (BEI): 400 ml of an X+7 culture was added with 40 ml of 2% (w/v) sodium bicarbonate to raise the pH to 7.5. An amount of 0.33 g of 2-bromoethylamine hydrobromide in 10 ml of deionized water was sterilized through a 0.2 µm filter and added to the culture. The culture was vortexed in situ at 37ºC for 24 hours (75 rpm). 10 ml of deionized water containing 0.5 g of sodium thiosulfate was filter sterilized and added to the inactivated culture to neutralize the BEI. The neutralized culture was vortexed at 37ºC for 24 hours (75 rpm).

To prepare the bacterins, the cells of each inactivated culture were centrifuged for 40 minutes at 15,000 xg and 4ºC, then resuspended in approximately 50 ml PBS and washed three times in this manner. The final pellet was resuspended in approximately 50 ml PBS. The resuspended Cells were mixed with Carbopol so that the final preparation contained Carbopol at a concentration of 0.2% (w/v) and comprised a dose of 2 ml of 5 x 10¹⁰ CCU. The vaccines also contained 0.005% (w/v) thimerosal as a preservative.

Piglets aged approximately one and three weeks were vaccinated intramuscularly in the neck region with a 2 ml dose of the appropriate bacterin. One group of pigs was not vaccinated and served as controls for the challenge test.

M. hyopneumoniae isolate 1002, X passage, was diluted to X+2 in the medium described above, using 10% inoculum at each time. The culture was vortexed at 75 rpm for 80 hours at 37ºC. At the end of the X+2 passage incubation, the amount of growth and the concentration of mycoplasma were determined in the same manner as the mycoplasma concentration for the vaccine. On the same day, these animals received the first vaccination, with several one-week-old piglets being inoculated with 2 ml of the above infectious culture. The inoculum was injected into the trachea through a 3 1/2 French Tom Cat catheter inserted through a 15-gauge needle. The infection was allowed to develop for three weeks.

The culture with which the test pigs were challenged was prepared as described above, however, the X+2 passage culture after 72, 96 and 120 hours of growth was used for exposure. At four weeks of age (one week after the second vaccination), unvaccinated and vaccinated animals were sedated with 1 to 2 ml of a solution of protnacevetalar (1:10). After sedation, a dose of 3 ml of the infectious culture was administered intranasally (1.5 ml per nostril) via a syringe connected to a short rubber tube inserted into the nostril. This procedure was repeated on the following two days using the same culture. In a sample of the 72-hour culture, the viable mycoplasma present were quantified by CCU. The titre was between 5 x 10⁸ and 5 x 10⁹ CCU/ml. At the time of exposure, Three to four carrier pigs (infected three weeks earlier) were mixed into each test group to serve as a natural source of infection.

Blood samples were collected from all piglets on the day of first vaccination (PV), on the day of second vaccination (Vi), on the first day of challenge (V2), one week after challenge (PCH1) and three weeks after challenge (PCH2). Serum from each blood sample was heat inactivated at 56ºC for 30 minutes and stored at -20ºC. Sera were tested for antibodies to M. hyopneumoniae by ELISA assay.

Twenty-one days after the first exposure, the animals were euthanized by injecting 2 to 5 ml of the euthanasia solution directly into the anterior vena cava. The thoracic cavities were immediately opened and the lungs removed whole. The lungs were photographed and large lesions were scored as follows. Ten points were scored for each apical lobe and each cardiac lobe, five points for the intermediate lobe, and five points for each outer edge of the diaphragmatic lobes, so that if all of these areas were completely densified (which would be an unusually severe case), the lesion score would be 55. The actual score obtained is then expressed as a percentage of this maximum score. This score is defined as the percent lesion score. The lesion scores were recorded on graphs.

The percentage lesion values were statistically analyzed by a two-tailed Student’s t-test to determine significant differences between the treatment groups.

The ELISA results are shown in Figure 1. At one week of age, before vaccination (PV), all groups had circulating antibodies to M. hyopneumoniae. These titers decreased at three weeks of age, before revaccination (Vi). At four weeks of age, before challenge (V2), animals vaccinated with either the formalin- or BEI-inactivated bacterin had higher titers than the unvaccinated animals. However, the formalin-inactivated bacterin produced dramatically higher titers than the BEI-inactivated bacterin.

The resulting lesion scores are shown in Table 3. The BEI-inactivated bacterin reduced the average percent lesion score by approximately 60%. This reduction is highly statistically significant (p < 0.01).

There appeared to be no relationship between the serological response and the percentage lesion scores. While formalin-inactivated bacterin elicited the highest titres of circulating antibodies, only BEI-inactivated bacterin protected pigs against infection.

These results suggest that circulating antibodies do not play the major role in immunological protection against Mycoplasma hyopneumoniae infection. Local secretory antibodies and/or cell-mediated immunity may be much more important.

The BEI-inactivated bacterin appears to properly activate these weapons of the immune system. Table 3 Percent lesion values of vaccinated pigs exposed to M. hyopneumoniae strain 1002

a. Not significantly different from non-vaccinated animals (p ) 0.5) according to the two-tailed T-test

b. Significantly different from non-vaccinated animals (p < 0.01) according to the two-sided t-test

Example 3

The Mycoplasma hyopneumoniae bacterin was investigated in a long-term immunity study. The bacterin was administered to pigs aged one to three weeks. Unvaccinated piglets from the litter served as controls. A portion of the vaccinated animals and the controls were exposed to virulent M. hyopneumoniae at the age of one month. A second group of vaccinated animals and controls were exposed to the virulent bacteria at two months of age. The remaining animals were exposed to the virulent bacteria at four months of age. Significant (p < 0.05) protection from infection was seen in vaccinated animals at one, two and four months of age. These results show that vaccination at one and three weeks of age provides long-lasting protection to pigs.

The bacterin used in this study contained 9.0 log₁₀ Mycoplasma hyopneumoniae DNA cell equivalents (MHDCE) per milliliter.

Seventy-six one-week-old piglets were divided into two treatment groups. Piglets from one litter were divided into the treatment groups. Sixty of these animals (30 per treatment group) were from Ionia Feeder Pigs, Inc., Ionia, Iowa. Sixteen animals (8 per treatment group) were from the SPF Swine Operation at Solvay Animal Health, Inc., Charles City, Iowa. The first treatment group of pigs was vaccinated at one week of age by intramuscular injection into the neck muscle behind the right ear. Injections were repeated two weeks later into the left side of the neck. The second treatment group of animals was not vaccinated.

The Ionia pigs were weaned at three weeks of age and placed in an isolation room. The SPF pigs remained with the SPF herd until exposure.

The infectious material was prepared and administered as described above. At one month of age, 10 vaccinated Ionia animals and 10 Ionia controls, plus 2 vaccinated SPF animals and 2 SPF controls were transferred to an isolation room and challenged with the infectious agent. At two months of age, 8 of the vaccinated Ionia animals and 8 controls, plus one vaccinated SPF animal and three SPF controls were transferred to an isolation room and challenged with the infectious agent. At four months of age, 8 of the vaccinated Ionia animals and 8 controls, plus 5 vaccinated SPF animals and 3 SPF controls were transferred to an isolation room and challenged with the infectious agent. Isolation room and brought into contact with the infectious agent.

Three weeks after challenge, all pigs were euthanized and percent lung lesions were determined as described above. Animals that died during the course of the study were not included in the analysis. A total of 8 non-vaccinated and 6 vaccinated animals died during the course of the study. These animals were generally in poor condition or suffered from severe intestinal inflammation, pleurisy or pericarditis. Animals that had large lung lesions at necropsy that were not caused by M. hyopneumoniae, i.e. surface fibrin, thoracic adhesions or hemorrhagic abscesses, were not included in the analysis. A total of one non-vaccinated animal and three vaccinated animals showed such lesions.

The percentage lung lesion values were subjected to a statistical analysis using the one-tailed t-test according to Student.

The results of the lesion scores and statistical analysis are presented in Table 4. There was an apparent age-dependent resistance to M. hyopneumoniae infection that developed in all pigs, including the unvaccinated animals. The average percent lesion scores in both vaccinated and unvaccinated animals decreased with the age of the animals at the time of challenge. It is unclear whether this increased resistance to infection is an immunological or a physiological phenomenon.

The vaccinated animals received significant (p < 0.05) protection even at four months of age. The actual percentage protection, calculated as described above, decreased with age. However, the 63 percent protection shown at four months of age still represents a significant reduction in lesions.

The results of this test demonstrate that the M. hyopneumoniae bacterin is capable of inducing long-lasting protection in pigs. Pigs vaccinated at one and three weeks of age are significantly protected, even up to four months of age. The average commercial slaughter pig lives for about 5 to 5.5 months. Vaccination with the M. hyopneumoniae bacterin should therefore provide protection during the crucial growth period. M. hyopneumoniae infections that only occur after four months of age should not significantly reduce performance. Table 4 Percent lesion values Age at exposure (months) Statistical analysis (vaccinated versus control, t-test, one-sided)

Example 4

In this example, Mycoplasma hyopneumoniae bacterins containing 7.0, 8.0, 9.0, 9.25, and 10.0 log10 M. hyopneumoniae DNA cell equivalents (MHDCE) per milliliter were evaluated in a minimum protective dose (MHP) study. The bacterins were administered as 2 ml intramuscular injections to pigs at one and three weeks of age. Significant (p < 0.05) protection against infection was elicited by bacterins containing ≥ 9. log10 MHDCE per milliliter.

The animals used in this study were from Ionia Feeder Pigs, Ionia, Iowa. 128 one-week-old pigs were divided into eight treatment groups of 16 pigs each. Piglets from one litter were distributed among the treatment groups. Pigs were marked in the ear for identification. Three groups were not vaccinated. Each of the other five groups received one of the five bacterins. The bacterins were administered as 2-mL intramuscular (i.m.) injections into the neck muscle behind the right ear. The injection was repeated two weeks later into the left side of the neck.

The animals were weaned at three weeks of age and transported to the research facilities of Solvay Animal Health, Inc. The vaccinated pigs and two of the unvaccinated groups were housed in two isolation rooms at Solvay. One of the unvaccinated groups was placed in each of the two isolation rooms. The third The unvaccinated group was housed separately to avoid contact with a foreign M. hyopneumoniae.

At four weeks of age, the vaccinated animals and the two groups of non-vaccinated animals (NV/CH) were challenged with virulent M. hyopneumoniae. The third non-vaccinated group (NV/NC), which was housed separately, was not challenged with M. hyopneumoniae.

During the experiment, serum samples were collected from each pig at 1, 3, 4 and 7 weeks of age. Antibodies to M. hyopneumoniae were quantified by ELISA as described in Example 2.

Three weeks after exposure, all pigs were euthanized and percentage lung lesions were determined. Animals with large lung lesions not caused by M. hyopneumoniae, i.e. surface fibrin, thoracic adhesions, or hemorrhagic abscesses, were not included in the analysis. Animals that died during the course of the study were not included in the analysis.

The percentage lung lesion values were statistically analyzed using a one-sided Student's t-test. The test showed that the two unvaccinated, exposed control groups did not differ significantly from each other. The two groups were therefore combined before the t-test analysis of the other treatment groups.

The ELISA results are shown in Table 5. At one week of age, the animals showed measurable M. hyopneumoniae antibodies. The ELISA uses whole-cell M. hyopneumoniae antigen. For this reason, it is difficult to exclude the possibility that some of these maternally derived antibodies are directed against Mycoplasma flocullare. M. hyopneumoniae and M. flocullare share some antigens. Nevertheless, these maternal antibodies appear to disappear rapidly and are consistently low by three weeks of age.

Those animals vaccinated with bacterins containing ≥ 9.0 log₁₀ MHDCE per milliliter showed seroconversion after the second vaccination at three weeks of age.

An unexpected result was the increased titer in the non-vaccinated, non-exposed control group at four weeks of age before any exposure. However, the increase was much less dramatic than in the groups of vaccinated animals.

These results suggest that bacterins containing ≥ 9.0 log₁₀ MHDCE per milliliter elicit effective titers of circulating antibodies against M. hyopneumoniae.

The results of the lesion scores and statistical analysis are presented in Table 6. To calculate the percentage protection, the average percentage lesion score of the unvaccinated, unexposed group was subtracted from the average percentage lesion score of the other treatment groups. The calculated mean values obtained were then inserted into the following formula:

It is clear that the percentage protection induced by vaccination increased with increasing dose. T-test analysis showed that significant (p < 0.05) protection was obtained with bacterins containing ≥ 9.0 log₁₀ MHDCE per milliliter.

The treatment group receiving bacterin containing 7.0 log₁₀ MHDCE per ml showed an average percent lesion score that was greater than the nonvaccinated, exposed control group, suggesting that suboptimal doses may increase the development of lesions after exposure.

These results lead to the conclusion that the minimal protective dose is approximately 2 x 10⁹ MHDCE (2 ml bacterin containing 9.0 log₁₀ MHDCE per ml). Table 5 Average values of ELISA

* no serum samples available Table 6 Percent lesion values

* NV/CH animals 1 to 13 were kept in a specific room during exposure, animals 14 to 28 were kept in a different room. Table 6 Statistical analysis (T-test, one-sided)

* Significantly different from the NV/CH group (p < 0.05)

Example 5

A fluorometric assay for measuring Mycoplasma hyopneumoniae using the DNA-binding dye Hoechst 33258 is described. The assay involves centrifugation of a M. hyopneumoniae culture to concentrate the cells, lysis of the cells in a detergent buffer, and assay of the lysate. A minifluorometer (TKO-100, Hoefer Scientific) is used and a standard curve is prepared using purified DNA determined spectrophotometrically. Growth of a M. hyopneumoniae culture is monitored using the assay and the results compared with the conventional assay using color-changing units (CCU).

One problem in the study of Mycoplasma hyopneumoniae is the difficulty in monitoring the growth of the organism. Because the efficiency of plating varies greatly at different stages of growth and because the colonies are small and grow extremely slowly, colony forming unit (CFU) tests cannot be used. Traditionally, M. hyopneumoniae cells have been counted using a color changing units (CCU) test. This test requires several dilutions of a culture to be made in tubes of fresh medium. The dilutions are then incubated for 14 days at 37ºC. Those tubes containing Mycoplasma hyopneumoniae show a pH shift to the acidic range. The titer of the original Culture is expressed as the value of the highest dilution that still shows growth. Because of the large experimental error in the CCU method and the large number of replicate tubes required for the test, there is a need for a faster and more accurate test.

The Hoechst 33258 dye and the DNA minifluorometer TKO 100 were purchased from Hoefer Scientific Products. Herring sperm DNA was from Sigma Chemical Co. All other chemicals were obtained from Sigma Chemical Co. in reagent grade.

M. hyopneumoniae isolate 1002 was grown in 200 ml cultures in shake flasks as described in Example 1 above. Six flasks were inoculated and incubated in parallel. Each culture was sampled at 24 hour intervals to measure pH and perform CCU and DNA assays. The pH values of each of the six cultures were calculated as an average for each time point.

The color changing assays were performed as described in Example 1 above. The average values of the CCU results from the six cultures were calculated for each time point.

For DNA testing, four 1.5 ml aliquots from each culture were placed in microfuge tubes and centrifuged at 12,000 x g for 10 minutes. The supernatants were completely removed and the pellets were resuspended in 120 µl of 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% (w/v) sodium dodecyl sulfate (TNES). The resuspended pellets were vigorously vortexed for 10 seconds. Ten µl of the suspension was mixed in a glass cuvette with 2 ml of the 0.04% (w/v) Hoechst dye solution in 10 mM Tris (pH 7.4), 150 mM NaCl and 1 mM EDTA. The cuvette was placed in a TKO 100 DNA minifluorometer, which had been previously calibrated using herring sperm DNA standards diluted in TNES. The values obtained from the four aliquots of each sample were averaged and the results from the six cultures were also averaged.

The dynamic linear response range of the assay was approximately 20 to 2000 ng DNA per 10 µl of test sample. This gave an actual range of 0.16 to 16 µg DNA per ml of culture. The genome of M. hyopneumoniae is approximately 5 x 10⁸ daltons (S. Razin, The Mycoplasmas, Microbiol. Rev. 42 (1978), 414- 470). This corresponds to 8 x 10⁻¹⁰ µg DNA per cell. The lower limit of the fluorometric assay is thus (0.16 µg DNA per ml of culture) ÷ (8 x 10⁻¹⁰ µg DNA per cell) = 2 x 10⁻ cells per ml of culture. The upper limit is 100 times this value or 2 x 10¹⁰ cells per ml.

This test measures cell concentration indirectly, so results are reported as M. hyopneumoniae DNA cell equivalents or MHDCE per milliliter.

During the logarithmic growth phase (24 to 72 hours), there is a good correlation between MHDCE and CCU values. MHDCE values are four to five times higher than CCU values. It is generally believed that the CCU test underestimates mycoplasma concentrations. The DNA test can therefore more accurately reflect the true number of M. hyopneumoniae cells. The CCU test is inherently susceptible to experimental error. To improve the accuracy of the CCF test, a large number of replicate tubes and better dilution series (two or four times) are required. The DNA test is less susceptible to experimental error. There is also some error in the DNA test, due to differences in sample handling and the measurement of very small volumes. However, this error depends on the skill of the technician performing this test. Most technicians can perform the DNA test very well with some practice and experience.

The DNA test measures all DNA, whether it is in living or dead cells or free in solution. The CCU test, on the other hand, measures only living cells. This explains why the MHDCE in a culture is slightly higher at 0 hours than at 24 hours. A significant portion of the DNA measured at 0 hours may be in dead or dying cells or in cells that do not survive serial transfer.

In stationary phase cultures (> 72 hours), the CCU test clearly shows a decrease in viable cells. Since M. hyopneumoniae is sensitive to acidic conditions, this death may be due to the pH. The DNA test is slower to respond, presumably because the dead or dying cells still have DNA that is detected.

The CCU test for the quantitative determination of M. hyopneumoniae has several disadvantages. First, it may underestimate the number of mycoplasma cells. Second, the test is prone to experimental errors. A large number of replica tubes, expensive medium and a lot of labor are needed to reduce these errors. Third, the test takes at least 14 days to complete.

The DNA test solves all of these problems. The estimated Mycoplasma cell counts are four to five times higher than the CCU counts, and they more accurately reflect the actual cell counts. With the right equipment and practice, the DNA test is less prone to error than the CCU test. Finally, the DNA test only takes 15 to 30 minutes to complete (a longer time is required if multiple samples are to be analyzed). For this reason, the DNA test is suitable for real-time monitoring of Mycoplasma cultures. However, it should be remembered that the DNA test measures all cells, whether they are alive or dead, and regardless of their metabolic state.

The test is therefore most meaningful during the log phase of the mycoplasma growth curve.

Claims (22)

1. A bacterin comprising an effective immunizing amount of a virulent Mycoplasma hyopneumoniae isolate 1002 (ATCC accession number 55088) inactivated with binary ethyleneimine at a concentration between 1 and 10 mM, in an amount of at least 10⁹ Mycoplasma hyopneumoniae DNA cell equivalents per ml of bacterin and a suitable physiologically acceptable carrier. 2. The bacterin of claim 1, further comprising an effective amount of an adjuvant. 3. Bacterin according to claim 2, wherein the adjuvant comprises a polymer of acrylic acid. 4. The bacterin of claim 3, wherein the effective amount of the adjuvant comprises about 0.2% (w/v) of the bacterin. 5. A process for producing the bacterin according to any one of claims 1 to 4, comprising (a) growing a virulent Mycoplasma hyopneumoniae isolate 1002 (ATCC accession number 55088) in culture on an appropriate medium; (b) treating the virulent Mycoplasma hyopneumoniae thus grown with binary ethyleneimine at a concentration of between 1 and 10 mM, thereby inactivating Mycoplasma hyopneumoniae; (c) Concentration of the resulting inactivated Mycoplasma hyopneumoniae; (d) obtaining the concentrated inactivated Mycoplasma hyopneumoniae; and (e) admixing a suitable physiologically acceptable carrier with the concentrated inactivated Mycoplasma hyopneumoniae thus obtained to formulate the bacterin. 6. The method of claim 5, wherein in step (a) the Mycoplasma hyopneumoniae is cultured for about 48 hours to about 144 hours. 7. A method according to claim 5 or 6, wherein in step (b) the treatment comprises the following steps: (i) contacting the Mycoplasma hyopneumoniae culture with binary ethyleneimine at a concentration of approximately 4 mM; (ii) incubating the culture under conditions effective to inactivate Mycoplasma hyopneumoniae; and (iii) Neutralization of the binary ethyleneimine in the culture by addition of sodium thiosulfate at an effective neutralizing concentration. 8. The method of claim 7, wherein in step (ii) the effective conditions comprise incubation for at least about 12 hours. 9. The method of claim 7 or 8, wherein in step (iii) the effective neutralizing concentration of sodium thiosulfate is about 16 mM. 10. A process according to any one of claims 7 to 9, wherein in step (i) the binary ethyleneimine which is contacted with Mycoplasma hyopneumoniae is generated in situ by adding L-bromoethylamine hydrobromide to the culture. 11. The method of any one of claims 5 to 10, wherein in step (e) the admixture further comprises admixing an effective amount of EDTA and an effective concentration of thimerosal. 12. The method of claim 11, wherein the effective concentration of EDTA comprises at least about 0.07% (w/v) of the bacterin. 10 13. The method of claim 11 or 12, wherein the effective concentration of thimerosal comprises about 0.01% (w/v) of the bacterin. 14. A method for inactivating a virulent strain of Mycoplasma hyopneumoniae isolate 1002 (ATCC accession number 55088), comprising: (a) contacting a culture of Mycoplasma hyopneumoniae with binary ethyleneimine at a concentration of approximately 4 mM; (b) incubating the culture under conditions effective to inactivate Mycoplasma hyopneumoniae; and (c) Neutralization of the binary ethyleneimine in the culture by addition of sodium at an effective neutralizing concentration. 15. The method of claim 14, wherein in step (b) the effective conditions comprise incubation for at least about 12 hours. 16. The method according to claim 14 or 15, wherein in step (c) the sodium is added in the form of sodium thiosulfate. 17. A method according to any one of claims 14 to 16, wherein in step (c) the effective neutralizing concentration of sodium thiosulfate is about 16 mM. 18. Bacterin according to one of claims 1 to 4 for immunizing a pig against infection by Mycoplasma hyopneumoniae. 19. Bacterin according to one of claims 1 to 4 or 18 for intramuscular injection. 20. Bacterin according to any one of claims 1 to 4, 18 or 19, wherein a dose comprises about 2 ml of the bacterin containing about 10⁹ Mycoplasma hyopneumoniae DNA cell equivalents per ml. 21. Bacterin according to any one of claims 1 to 4 or 18 to 20, which can be administered to the pig twice; once about one week and once about three weeks after the birth of the pig. 22. Virulent Mycoplasma hyopneumoniae isolate designated 1002 (ATCC accession number 55088) inactivated with binary ethyleneimine.